This work is directed towards an understanding, on the molecular level, of the mechanism by which Na ion and K ion are actively moved across the plasma membranes of animal cells by the enzyme Na,K-ATPase, fueled by the breakdown of ATP. The Na,K-ATPase is now thought to be made up of a number of subunits, including a dimer of large polypeptides that catalyze the hydrolysis of ATP. In this work I propose to study two questions regarding subunit interactions as they relate to mechanism: 1) Are the large subunits structurally and functionally identical? 2) How do the large subunits interact with regard to the binding of ATP, phosphorylation, and the binding of cardiac glycosides; how does this interaction change at different points in the transport cycle? The approach relies on a novel technique whereby one polypeptide chain of the dimer is singled out by covalently labeling it with a radioactive photoaffinity derivative of ouabain and isolating it using an antibody to ouabain. This chain is then analyzed to see (1) if it is the same as those chains that are not labeled, or (2) to determine what other radioactive ligands (e.g., ATP, phosphate, group specific reagents) are bound to the ouabain labeled chain or to the other chain. The interaction of the two polypeptides as regards ligand binding sites at different stages in the transport cycle should provide insight into the molecular mechanism by which Na and K are moved across the membrane.